The invention relates to detection and/or quantification of nucleic acid sequences using nucleic acid detector probes.
The development of nucleic acid amplification methods contributed to the rapid advances in genome research, biotechnology, molecular medicine, and the like. Rapid methods for the detection and quantification of specific nucleic acid sequences, either directly or following amplification, are required for nucleic acid analysis as used in various research, clinical and industrial applications. Various homogenous or heterogeneous methods for the detection of binding of the one or more labels to the target sequence were described.
Nucleic acid hybridization is commonly used for the detection of specific sequences. Methods for the detection and quantification of nucleic acid sequences, which utilize hybridization of one or more oligonucleotide probes to a specific target sequence, usually employ means of detection of hybrid formation. Some methods utilize one or more labeled probes that are complementary to a specific region of the target nucleic acid sequence, and means of detecting binding of the one or more labels to the target sequence.
Some of the previously described detection methods employ enzyme-catalyzed reactions. These methods are based on detection of the formation of hybrids of the specific target nucleic acid and one or more oligonucleotide probes, which may be labeled. Both enzymatic cleavage and ligation of specific oligonucleotide probes hybridized to target nucleic acid sequence have been described. The enzymatic reaction products are detected directly, for example, by changes of optical properties of the labels, or by their capacity to induce additional enzymatic reaction or become substrates for additional enzymes. The enzyme-based detection methods may be employed either for the direct detection of specific nucleic acid sequences, or may be employed for the detection of amplification products. Some of the commonly used homogeneous detection methods employing enzymes which cleave oligonucleotide probes when bound to the target nucleic acid sequence by hybridization, are CYCLING PROBE METHOD(trademark), TAQMAN(trademark), INVADER(trademark), and the like. Many of these methods employ an oligonucleotide which is labeled with a pair of a fluorescent and a quencher labels, which are attached to the probe at the correct distance to ensure energy transfer from the fluorescent label to the quencher label. Cleavage of one of the probes when hybridized to the target nucleic acid sequence results in separation of the two labels which results in the generation of fluorescent signal. Various modifications of this principle have been described (Nurmi et al., 2000, Nucleic acid Res. 28, 28e; U.S. Pat. No. 5,403,711 (Walder); Nadeau et al., 1999, Anal Biochem 276: 177).
Other methods employ means of direct, non-enzymatic, detection of one or more probe-target hybrid formation. Both homogeneous and heterogeneous detection methods have been described. Heterogeneous detection methods that require extensive wash steps are inherently more complicated and are less desirable. Homogeneous detection methods are faster and are better suited for applications requiring high throughput. Hybridization of one or more labeled probes to a specific target nucleic acid sequence may result in changes in the optical properties of the one or more labeled probes. Alternatively, the association of two or more labels in a stable complex, as a result of hybridization of two or more oligonucleotide probes to a single nucleic acid target, can be detected.
Homogeneous detection of hybrid formation based on the separation of donor-acceptor label molecules has been described recently. A method utilizing a change in probe conformation upon hybridization to a target nucleic acid sequence thereby changing the distance of donor acceptor label molecules, was described by Tyagi et al. (U.S. Pat. No. 5,925,517). The probe is composed of a stem loop structure, which brings the donor-acceptor pair of labels attached to the probe in close proximity to effect fluorescence quenching. Hybridization of the probe to a specific target nucleic acid sequence results in a conformational change which is characterized by dissociation of the stem structure thereby increasing the distance between the two labels and preventing energy transfer between the two labels. Thus, hybridization of the oligonucleotide to the specific target nucleic acid sequence is detectable by a fluorescence signal of the donor label. Other variations on this concept have also been disclosed. Detection of nucleic acid by fluorescence quenching was also described by Bruce et al (EP patent application no. 98117883.3). The detector oligonucleotide probe comprises at least two oligonucleotides, which hybridize to form a partially double stranded detector. Acceptor and donor dyes are attached to the detector oligonucleotides so as to place them at close proximity to enable energy transfer. Upon hybridization of the single stranded portion of the detector probe to the target nucleic acid sequence the second oligonucleotide probe is displaced from the first oligonucleotide probe, thus increasing the distance between the donor and acceptor dye labels, causing a change in fluorescence that is detectable.
There is a need for improved nucleic acid detection and/or quantification methods. The invention provided herein fulfills this need and also provides additional benefits. These include the ease of preparation of the detector probes. The design of the interacting sequences of the detector probes are independent of the target sequence and could thus be universal. Insofar as the detector probes are not unimolecular, the design complexities of a stem loop probe are eliminated.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.
In one aspect of the present invention, detector oligonucleotide probes which are useful for the detection of a specific nucleic acid sequence are provided. A first oligonucleotide probe comprises a 3xe2x80x2-region that is hybridizable to a sequence of the target nucleic acid; a 5xe2x80x2-region that is not hybridizable to the target nucleic acid (under otherwise same hybridization conditions as described herein), and a label (F) that is attached to the 5xe2x80x2-end. A second oligonucleotide probe comprises a 5xe2x80x2-region, which is hybridizable to a sequence of the target nucleic acid and a 3xe2x80x2-region, which is not hybridizable to the target nucleic acid (under otherwise same hybridization conditions as described herein), and is hybridizable to part of the sequence of the first oligonucleotide which is not hybridizable to the target nucleic acid. A third oligonucleotide probe comprises a sequence which is not hybridizable to the target nucleic acid sequence and is hybridizable to the 5xe2x80x2-most sequence of the first oligonucleotide, and a label Q that is attached to its 3xe2x80x2-end. In some embodiments, probes with the mirror image design of the preceding oligonucleotide probes are provided. In some embodiments, a single oligonucleotide probe comprises both labels and the functions of either the first and third oligonucleotide probe, or the second and third oligonucleotide probe. Mirror images (i.e., the opposite polarity) of the probes are also provided.
In another aspect of the present invention, methods for detecting and/or quantifying nucleic acid sequences are provided using the probes described above. Detection of a target nucleic acid according to the methods of the invention comprises a) combining a sample suspected of containing said target nucleic acid sequence with a mixture containing the probes described above; optionally b) treating the mixture to render the target nucleic acid single stranded (if not already single stranded); and c) incubating the mixture under conditions which are suitable for binding of the oligonucleotide probes to said single stranded nucleic acid target, wherein binding of the first and second probes to the nucleic acid target results in the displacement of the third probe from the first probe, wherein said displacement results in the spatial separation of the labels of the first probe and the third probe, whereby a detectable and/or quantifiable signal is generated.
In another aspect of the present invention, a method of determining whether a target nucleic acid sequence is present in a sample is provided, comprising: contacting said sample with a first probe, a second probe, and a third probe, under conditions allowing hybridization of the first and second probes to the target nucleic acid sequence, if present, and hybridization of the first probe to the third probe, in the absence of the target nucleic acid sequence, and hybridization of the second probe to the first probe when the second probe is hybridized to the target nucleic acid sequence, wherein: the first probe comprises a polynucleotide comprising a 3xe2x80x2 region which hybridizes to a first region of the target nucleic acid sequence, if present, and a first member of an interacting label pair; the second probe comprises a polynucleotide comprising a 5xe2x80x2 region which hybridizes to a second region of the target nucleic acid sequence, if present, and a 3xe2x80x2 region which hybridizes to a sequence in the 5xe2x80x2 region of the first probe, if the target nucleic acid sequence is present; and the third probe comprises a polynucleotide which hybridizes to a sequence in the 5xe2x80x2 region of the first probe, and a second member of an interacting label pair; wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact; and wherein, in the presence of the target nucleic acid sequence, said 3xe2x80x2 region of said first probe hybridizes to said first region of the target nucleic acid sequence and said 5xe2x80x2 region of said second probe hybridizes to said second region of the target nucleic acid sequence and said 3xe2x80x2 region of said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair; whereby generation of a detectable signal caused by dissociation of the interacting label pair indicates presence of the target nucleic acid sequence. This aspect may further comprise extension of the 3xe2x80x2 end of the second probe along a sequence in the 5xe2x80x2 region of the first probe using a nucleotide polymerase.
In a further aspect of the present invention is provided a method of determining whether a target nucleic acid sequence is present in a sample, comprising: contacting said sample with a first probe, a second probe, and a third probe, under conditions allowing hybridization of the first and second probes to the target nucleic acid sequence, if present, and hybridization of the first probe to the third probe, in the absence of the target nucleic acid sequence, and hybridization of the second probe to the first probe when the second probe is hybridized to the target nucleic acid sequence, wherein the first probe comprises a polynucleotide comprising a 5xe2x80x2 region which hybridizes to a first region of the target nucleic acid sequence, if present, and a first member of an interacting label pair; the second probe comprises a polynucleotide comprising a 3xe2x80x2 region which hybridizes to a second region of the target nucleic acid sequence, if present, and a 5xe2x80x2 region which hybridizes to a sequence in the 3xe2x80x2 region of the first probe, if the target nucleic acid sequence is present; and the third probe comprises a polynucleotide which hybridizes to a sequence in the 3xe2x80x2 region of the first probe, and a second member of an interacting label pair; wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact; and wherein, in the presence of the target nucleic acid sequence, said 5xe2x80x2 region of said first probe hybridizes to said first region of the target nucleic acid sequence and said 3xe2x80x2 region of said second probe hybridizes to said second region of the target nucleic acid sequence and said 5xe2x80x2 region of said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair; whereby generation of a detectable signal caused by dissociation of the interacting label pair indicates presence of the target nucleic acid sequence.
In the methods of the invention, the first and second regions of the target nucleic acid sequence may be contiguous, 1 nucleotide apart, 3 or fewer nucleotides apart, or 7 or fewer nucleotides apart. In addition, the interacting label pair may comprise donor and acceptor moieties, or the interacting label pair may comprises an enzyme, such as an enzyme and an inhibitor of said enzyme, or the interacting label pair may be capable of energy transfer, for example the interacting label pair may comprise a fluorophore and a quencher.
The methods may further comprise measuring the magnitude of the signal generated, whereby said magnitude indicates the quantity of the target nucleic acid sequence.
The target nucleic acid sequence may be attached to an analyte, or to a solid support. The target nucleic acid sequence may comprise DNA, RNA, DNA and RNA, or PNA. At least one of the probes may comprise DNA, RNA, DNA and RNA or PNA. The region of the second probe which is hybridizable to a sequence of the first probe may comprise a modified nucleotide that causes enhanced affinity to the sequence in the region of the first probe relative to an unmodified nucleotide. In one aspect, the detectable signal is of a greater magnitude than a detectable signal associated with the interacting label pair when the third probe is hybridized to the first probe. In another aspect, the detectable signal is of a lesser magnitude than a detectable signal associated with the interacting label pair when the third probe is hybridized to the first probe. In a further aspect, the method further comprises amplifying the target nucleic acid sequence. In yet a further aspect, the first probe or the second probe is allele-specific. In a still further aspect, the first probe and the second probe are allele-specific.
In a further aspect, the invention provides a method of determining whether a target nucleic acid sequence is present in a sample, comprising: contacting said sample with a first probe, a second probe, a third probe, and a nucleotide polymerase, under conditions allowing hybridization of the first and second probes to the target nucleic acid sequence, if present, and hybridization of the first probe to the third probe, in the absence of the target nucleic acid sequence, and allowing nucleic acid polymerization, wherein: the first probe comprises a polynucleotide comprising a 3xe2x80x2 region which hybridizes to a first region of the target nucleic acid sequence, if present, a first member of an interacting label pair; and the second probe comprises a polynucleotide comprising a 5xe2x80x2 region which hybridizes to a second region of the target nucleic acid sequence, if present; and the third probe comprises a polynucleotide sequence which hybridizes to a sequence in the 5xe2x80x2 region of the first probe, and a second member of an interacting label pair; wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact; and wherein, in the presence of the target nucleic acid sequence, said 3xe2x80x2 region of said first probe hybridizes to said first region of the target nucleic acid sequence and said 5xe2x80x2 region of said second probe hybridizes to said second region of the target nucleic acid sequence; and wherein the 3xe2x80x2 end of said second probe is extended by said nucleotide polymerase along the first probe by template switching, causing dissociation of the first and second members of the interacting label pair; whereby generation of detectable signal caused by dissociation of the interacting label pair indicates presence of the target nucleic acid sequence.
In a further aspect, the invention provides a method of determining whether a target nucleic acid sequence is present in a sample, said method comprising: contacting said sample with a first probe and a second probe, under conditions allowing hybridization of the first and second probes to the target nucleic acid sequence, if present, and hybridization of a first nucleotide sequence in the 5xe2x80x2 region of the first probe with a second nucleotide sequence in the 5xe2x80x2 region of the first probe, in the absence of the target nucleic acid sequence, and hybridization of at the second probe the first probe when the second probe is hybridized to the target nucleic acid sequence, wherein: the first probe comprises a polynucleotide comprising a 3xe2x80x2 region which hybridizes to a first region of the target nucleic acid sequence, if present, and a 5xe2x80x2 region comprising said first and second nucleotide sequences which hybridize to each other in the absence of the target nucleic acid sequence, and two members of an interacting label pair; the second probe comprises a polynucleotide comprising a 5xe2x80x2 region which hybridizes to a second region of the target nucleic acid sequence, if present and a 3xe2x80x2 region which hybridizes to a sequence in the 5xe2x80x2 region of the first probe, if the target polynucleotide is present; wherein, when said first and second nucleotide sequences in the 5xe2x80x2 region of the first probe hybridize, said first and second members of the interacting label pair are brought into proximity and interact; and wherein, in the presence of the target nucleic acid sequence, said first probe and said second probe hybridize to the target nucleic acid sequence and said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair; whereby generation of detectable signal caused by dissociation of the interacting label pair indicates presence of the target nucleic acid sequence.
In yet a further aspect, the invention provides a method of determining whether a target nucleic acid sequence is present in a sample, said method comprising: contacting said sample with a first probe and a second probe, under conditions allowing hybridization of the first and second probes to the target nucleic acid sequence, if present, and hybridization of a first nucleotide sequence in the 3xe2x80x2 region of the first probe with a second nucleotide sequence in the 3xe2x80x2 region of the first probe, in the absence of the target nucleic acid sequence, and hybridization of at the second probe the first probe when the second probe is hybridized to the target nucleic acid sequence, wherein: the first probe comprises a polynucleotide comprising a 5xe2x80x2 region which is hybridizes to a first region of the target nucleic acid sequence, if present, and a 3xe2x80x2 region comprising said first and second nucleotide sequences which hybridize to each other in the absence of the target nucleic acid sequence, and two members of an interacting label pair; the second probe comprises a polynucleotide comprising a 3xe2x80x2 region which hybridizes to a second region of the target nucleic acid sequence, if present and a 5xe2x80x2 region which hybridizes to a sequence in the 3xe2x80x2 region of the first probe, if the target polynucleotide is present; wherein, when said first and second nucleotide sequences in the 3xe2x80x2 region of the first probe hybridize, said first and second members of the interacting label pair are brought into proximity and interact; and wherein, in the presence of the target nucleic acid sequence, said first probe and said second probe hybridize to the target nucleic acid sequence and said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair; whereby generation of detectable signal caused by dissociation of the interacting label pair indicates presence of the target nucleic acid sequence.
In yet another aspect of the present invention, methods for detecting changes, or mutations, in a sequence of the target nucleic acid relative to a reference nucleic acid sequence (unaltered target sequence), are provided. In one embodiment, detection of altered sequence in a target nucleic acid according to the methods of the invention comprises a) combining a test sample suspected of containing said target nucleic acid sequence (with suspected altered target sequence) with a mixture containing the probes described above, wherein the first and/or third oligonucleotide probes comprise a sequence hybridizable to the unaltered (reference) target sequence; optionally b) treating the mixture to render the target nucleic acid single stranded (if not already single stranded); and c) incubating the mixture under conditions which are suitable for binding of the oligonucleotide probes to said single stranded nucleic acid target, wherein binding of the first and second probes to the nucleic acid target results in the displacement of the third probe from the first probe, wherein said displacement results in the spatial separation of the labels of the first probe and the third probe, whereby a detectable and/or quantifiable signal is generated. In this embodiment, reduction or elimination of detectable signal in the test sample (relative to a reference sample comprising unaltered target (reference) sequence) indicates reduced or no binding of the probe(s) to the nucleic acid target in the test sample, which indicates presence of the altered sequence in the test sample. In another embodiment, detection of altered sequence in a target nucleic acid according to the methods of the invention comprises a) combining a test sample suspected of containing said target nucleic acid sequence (with suspected altered target sequence) with a mixture containing the probes described above, wherein the first and/or third oligonucleotide probes comprise a sequence hybridizable to the suspected altered target sequence; optionally b) treating the mixture to render the target nucleic acid single stranded (if not already single stranded); and c) incubating the mixture under conditions which are suitable for binding of the oligonucleotide probes to said single stranded nucleic acid target, wherein binding of the first and second probes to the nucleic acid target results in the displacement of the third probe from the first probe, wherein said displacement results in the spatial separation of the labels of the first probe and the third probe, whereby a detectable and/or quantifiable signal is generated. In this embodiment, a greater amount of detectable signal in the test sample (relative to a reference sample comprising unaltered (reference) sequence) indicates more binding of the probe(s) to the nucleic acid target in the test sample, which indicates presence of the altered sequence in the test sample.
A further aspect of the present invention provides a method of determining whether a target nucleic acid sequence contains a sequence alteration relative to a reference nucleic acid sequence, said method comprising: contacting said target nucleic acid sequence with a first probe, a second probe, and a third probe, and contacting said reference nucleic acid sequence with a first probe, a second probe, and a third probe, wherein said contact of the target nucleic acid sequence with the first, second, and third probes, and said contact of the reference nucleic acid sequence with the first, second, and third probes, occur under conditions allowing hybridization of the first and second probes to the reference nucleic acid sequence, and hybridization of the first probe to the third probe, in the absence of the reference nucleic acid sequence, and hybridization of the second probe to the first probe when the second probe is hybridized to the reference nucleic acid sequence, wherein the first probe comprises a polynucleotide comprising a 3xe2x80x2 region which hybridizes to a first region of the reference nucleic acid sequence, if present, and a first member of an interacting label pair; the second probe comprises a polynucleotide comprising a 5xe2x80x2 region which hybridizes to a second region of the reference nucleic acid sequence, if present, and a 3xe2x80x2 region which hybridizes to a sequence in the 5xe2x80x2 region of the first probe, if the reference nucleic acid sequence is present; and the third probe comprises a polynucleotide which hybridizes to a sequence in the 5xe2x80x2 region of the first probe, and a second member of an interacting label pair; wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact; and wherein, in the presence of the reference nucleic acid sequence, said 3xe2x80x2 region of said first probe hybridizes to said first region of the reference nucleic acid sequence and said 5xe2x80x2 region of said second probe hybridizes to said second region of the reference nucleic acid sequence and said 3xe2x80x2 region of said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair; and comparing detectable signal generated by contacting said first, second, and third probes with the reference nucleic acid sequence with detectable signal generated by contacting said first, second, and third probes with target nucleic acid sequence; whereby reduced signal generation by the target nucleic acid sequence as compared to the reference nucleic acid sequence indicates the presence of an altered sequence in the target nucleic acid sequence relative to the reference nucleic acid sequence.
In yet a further aspect, the present invention provides a method of determining whether a target nucleic acid sequence contains a sequence alteration relative to a reference nucleic acid sequence, said method comprising: contacting said target nucleic acid sequence with a first probe, a second probe, and a third probe, and contacting said reference nucleic acid sequence with a first probe, a second probe, and a third probe, wherein said contact of the target nucleic acid sequence with the first, second, and third probes, and said contact of the reference nucleic acid sequence with the first, second, and third probes, occur under conditions allowing hybridization of the first and second probes to the reference nucleic acid sequence, and hybridization of the first probe to the third probe, in the absence of the reference nucleic acid sequence, and hybridization of the second probe to the first probe when the second probe is hybridized to the reference nucleic acid sequence, wherein the first probe comprises a polynucleotide comprising a 5xe2x80x2 region which hybridizes to a first region of the reference nucleic acid sequence, if present, and a first member of an interacting label pair; the second probe comprises a polynucleotide comprising a 3xe2x80x2 region which hybridizes to a second region of the reference nucleic acid sequence, if present, and a 5xe2x80x2 region which hybridizes to a sequence in the 3xe2x80x2 region of the first probe, if the reference nucleic acid sequence is present; and the third probe comprises a polynucleotide which hybridizes to a sequence in the 5xe2x80x2 region of the first probe, and a second member of an interacting label pair; wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact; and wherein, in the presence of the reference nucleic acid sequence, said 5xe2x80x2 region of said first probe hybridizes to said first region of the reference nucleic acid sequence and said 3xe2x80x2 region of said second probe hybridizes to said second region of the reference nucleic acid sequence and said 5xe2x80x2 region of said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair; and comparing detectable signal generated by contacting said first, second, and third probes with the reference nucleic acid sequence with detectable signal generated by contacting said first, second, and third probes with target nucleic acid sequence; whereby reduced signal generation by the target nucleic acid sequence as compared to the reference nucleic acid sequence indicates the presence of an altered sequence in the target nucleic acid sequence relative to the reference nucleic acid sequence.
As is evident to one skilled in the art, in these methods binding of first and second probes to the target nucleic acid sequence competes with binding of the third probe to the second probe, thus maintaining the spatial separation of the labels of the first probe and the third probe, whereby a detectable and/or quantifiable signal is generated .
In another aspect of the present invention, methods for genotype determination are provided. In these methods, the first and/or third oligonucleotide probes that are allele-specific are used.
Another aspect of the invention provides methods of detection and/or quantification of multiple target nucleic acid sequences in a sample. In these methods, two or more combinations (pairs) of interacting labels, each pair specific for a defined target nucleic acid sequence, are used.
Accordingly, the invention provides a method of determining whether one or more of a plurality of target nucleic acid sequences is present in a sample, comprising: contacting said sample with a plurality of probe sets, each set comprising a first probe, a second probe, and a third probe, under conditions allowing hybridization of the first and second probes to a target nucleic acid sequence, if present, and hybridization of the first probe to the third probe, in the absence of said target nucleic acid sequence, and hybridization of the second probe to the first probe when the second probe is hybridized to said target nucleic acid sequence, wherein the first probe comprises a polynucleotide comprising a 3xe2x80x2 region which hybridizes to a first region of said target nucleic acid sequence, if present, and a first member of an interacting label pair; the second probe comprises a polynucleotide comprising a 5xe2x80x2 region which hybridizes to a second region of said target nucleic acid sequence, if present, and a 3xe2x80x2 region which hybridizes to a sequence in the 5xe2x80x2 region of the first probe, if said target nucleic acid sequence is present; the third probe comprises a polynucleotide which hybridizes to a sequence in the 5xe2x80x2 region of the first probe, and a second member of an interacting label pair; wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact; and wherein, in the presence of said target nucleic acid sequence, said 3xe2x80x2 region of said first probe hybridizes to said first region of said target nucleic acid sequence and said 5xe2x80x2 region of said second probe hybridizes to said second region of said target nucleic acid sequence and said 3xe2x80x2 region of said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair; whereby generation of a detectable signal caused by dissociation of the interacting label pair indicates presence of said target nucleic acid sequence; and wherein each probe set comprises an interacting label pair which generates a detectable signal which is different from the signals of the interacting label pairs of every other probe set, and generation of two or more signals indicates presence of a plurality of target nucleic acid sequences.
In another aspect, the invention provides a method of determining whether one or more of a plurality of target nucleic acid sequences is present in a sample, comprising: contacting said sample with a plurality of probe sets, each set comprising a first probe, a second probe, and a third probe, under conditions allowing hybridization of the first and second probes to a target nucleic acid sequence, if present, and hybridization of the first probe to the third probe, in the absence of said target nucleic acid sequence, and hybridization of the second probe to the first probe when the second probe is hybridized to said target nucleic acid sequence, wherein the first probe comprises a polynucleotide comprising a 5xe2x80x2 region which hybridizes to a first region of said target nucleic acid sequence, if present, and a first member of an interacting label pair; the second probe comprises a polynucleotide comprising a 3xe2x80x2 region which hybridizes to a second region of said target nucleic acid sequence, if present, and a 5xe2x80x2 region which hybridizes to a sequence in the 3xe2x80x2 region of the first probe, if said target nucleic acid sequence is present; the third probe comprises a polynucleotide which hybridizes to a sequence in the 3xe2x80x2 region of the first probe, and a second member of an interacting label pair; wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact; and wherein, in the presence of said target nucleic acid sequence, said 5xe2x80x2 region of said first probe hybridizes to said first region of said target nucleic acid sequence and said 3xe2x80x2 region of said second probe hybridizes to said second region of said target nucleic acid sequence and said 5xe2x80x2 region of said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair; whereby generation of a detectable signal caused by dissociation of the interacting label pair indicates presence of said target nucleic acid sequence; and wherein each probe set comprises an interacting label pair which generates a detectable signal which is different from the signals of the interacting label pairs of every other probe set, and generation of two or more signals indicates presence of a plurality of target nucleic acid sequences.
The invention also provides compositions, kits, complexes, reaction mixtures and systems comprising various components (and various combinations of the components) used in the methods described herein.
In one aspect, the invention provides a composition comprising a first probe, a second probe and a third probe, wherein the first probe comprises a polynucleotide comprising a 3xe2x80x2 region which hybridizes to a first region of a target nucleic acid sequence, if present, and a first member of an interacting label pair; the second probe comprises a polynucleotide comprising a 5xe2x80x2 region which hybridizes to a second region of the target nucleic acid sequence, if present, and a 3xe2x80x2 region which hybridizes to a sequence in the 5xe2x80x2 region of the first probe, if the target nucleic acid sequence is present; the third probe comprises a polynucleotide which hybridizes to a sequence in the 5xe2x80x2 region of the first probe, and a second member of an interacting label pair; wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact; and wherein, in the presence of the target nucleic acid sequence, said 3xe2x80x2 region of said first probe hybridizes to said first region of the target nucleic acid sequence and said 5xe2x80x2 region of said second probe hybridizes to said second region of the target nucleic acid sequence and said 3xe2x80x2 region of said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair; whereby generation of a detectable signal caused by dissociation of the interacting label pair indicates presence of the target nucleic acid sequence.
In another aspect, the invention provides a composition comprising a first probe, a second probe and a third probe, wherein the first probe comprises a polynucleotide comprising a 5xe2x80x2 region which hybridizes to a first region of a target nucleic acid sequence, if present, and a first member of an interacting label pair; the second probe comprises a polynucleotide comprising a 3xe2x80x2 region which hybridizes to a second region of the target nucleic acid sequence, if present, and a 5xe2x80x2 region which hybridizes to a sequence in the 3xe2x80x2 region of the first probe, if the target nucleic acid sequence is present; the third probe comprises a polynucleotide which hybridizes to a sequence in the 3xe2x80x2 region of the first probe, and a second member of an interacting label pair; wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact; and wherein, in the presence of the target nucleic acid sequence, said 5xe2x80x2 region of said first probe hybridizes to said first region of the target nucleic acid sequence and said 3xe2x80x2 region of said second probe hybridizes to said second region of the target nucleic acid sequence and said 5xe2x80x2 region of said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair; whereby generation of a detectable signal caused by dissociation of the interacting label pair indicates presence of the target nucleic acid sequence.
The compositions may comprise an interacting moiety pair which comprises a fluorophore and a quencher. The compositions may further comprise a nucleotide polymerase the target nucleic acid sequence, and/or a reference nucleic acid sequence to which the target nucleic acid sequence is to be compared.
In another aspect, the invention provides kits for conducting the methods described herein. These kits, in suitable packaging and generally (but not necessarily) containing suitable instructions, contain one or more components used in the detection and/or quantification methods.
In one aspect, the invention provides a kit for determining whether a target nucleic acid is present in a sample or quantifying a target nucleic acid sequence, comprising a first probe, a second probe and a third probe, wherein the first probe comprises a polynucleotide comprising a 3xe2x80x2 region which hybridizes to a first region of a target nucleic acid sequence, if present, and a first member of an interacting label pair; the second probe comprises a polynucleotide comprising a 5xe2x80x2 region which hybridizes to a second region of the target nucleic acid sequence, if present, and a 3xe2x80x2 region which hybridizes to a sequence in the 5xe2x80x2 region of the first probe, if the target nucleic acid sequence is present; and the third probe comprises a polynucleotide which hybridizes to a sequence in the 5xe2x80x2 region of the first probe, and a second member of an interacting label pair; wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact; and wherein, in the presence of the target nucleic acid sequence, said 3xe2x80x2 region of said first probe hybridizes to said first region of the target nucleic acid sequence and said 5xe2x80x2 region of said second probe hybridizes to said second region of the target nucleic acid sequence and said 3xe2x80x2 region of said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair; whereby generation of a detectable signal caused by dissociation of the interacting label pair indicates presence of the target nucleic acid sequence.
In a further aspect, the invention provides a kit for determining whether a target nucleic acid is present in a sample or quantifying a target nucleic acid sequence, comprising a first probe, a second probe and a third probe, wherein the first probe comprises a polynucleotide comprising a 5xe2x80x2 region which hybridizes to a first region of a target nucleic acid sequence, if present, and a first member of an interacting label pair; the second probe comprises a polynucleotide comprising a 3xe2x80x2 region which hybridizes to a second region of the target nucleic acid sequence, if present, and a 5xe2x80x2 region which hybridizes to a sequence in the 3xe2x80x2 region of the first probe, if the target nucleic acid sequence is present; the third probe comprises a polynucleotide which hybridizes to a sequence in the 3xe2x80x2 region of the first probe, and a second member of an interacting label pair; wherein when said third probe is hybridized to said first probe, said first and second members of the interacting label pair are brought into proximity and interact; and wherein, in the presence of the target nucleic acid sequence, said 5xe2x80x2 region of said first probe hybridizes to said first region of the target nucleic acid sequence and said 3xe2x80x2 region of said second probe hybridizes to said second region of the target nucleic acid sequence and said 5xe2x80x2 region of said second probe hybridizes to the first probe, causing dissociation of the first and second members of the interacting label pair; whereby generation of a detectable signal caused by dissociation of the interacting label pair indicates presence of the target nucleic acid sequence.
Kits of the invention may further comprise a reference nucleic acid sequence to which the target nucleic acid sequence may be compared, and/or instructions for use of the kit to determine the presence of the target nucleic acid sequence in a sample or quantify the target nucleic acid sequence.
In another aspect, the invention provides compositions comprising any of the complexes (which are generally considered as intermediates with respect to the final products) described herein (see also the figures for schematic depictions of these various complexes).
In another aspect, the invention provides reaction mixtures (or compositions comprising reaction mixtures) which contain various combinations of components described herein.
In another aspect, the invention provides systems for effecting the detection and/or quantification methods described herein.